The present invention relates generally to methods for regulating occludin-mediated processes, and more particularly to the use of modulating agents comprising an occludin cell adhesion recognition sequence and/or an antibody that specifically recognizes such a sequence for inhibiting functions such as cell adhesion and the formation of tissue permeability barriers.
Cell adhesion is a complex process that is important for maintaining tissue integrity and generating physical and permeability barriers within the body. All tissues are divided into discrete compartments, each of which is composed of a specific cell type that adheres to similar cell types. Such adhesion triggers the formation of intercellular junctions (i.e., readily definable contact sites on the surfaces of adjacent cells that are adhering to one another), also known as tight junctions, gap junctions, spot desmosomes and belt desmosomes. The formation of such junctions gives rise to physical and permeability barriers that restrict the free passage of cells and other biological substances from one tissue compartment to another. For example, the blood vessels of all tissues are composed of endothelial cells. In order for components in the blood to enter a given tissue compartment, they must first pass from the lumen of a blood vessel through the barrier formed by the endothelial cells of that vessel. Similarly, in order for substances to enter the body via the gut, the substances must first pass through a barrier formed by the epithelial cells of that tissue. To enter the blood via the skin, both epithelial and endothelial cell layers must be crossed.
Cell adhesion is mediated by specific cell surface adhesion molecules (CAMs). There are many different families of CAMs, including the immunoglobulin, integrin, selectin and cadherin superfamilies, and each cell type expresses a unique combination of these molecules. Cadherins are a rapidly expanding family of calcium-dependent CAMs (Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co.(Austin Tex., 1996). The cadherins (abbreviated CADs) are membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell). Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. For example, N (neural)xe2x80x94cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. E (epithelial)xe2x80x94cadherin is predominantly expressed by epithelial cells. VE (vascular endothelial)xe2x80x94cadherin is predominantly expressed by endothelial cells. Other CADs are P (placental)xe2x80x94cadherin, which is found in human skin, and R (retinal)xe2x80x94cadherin. A detailed discussion of the cadherins is provided in Munro S B et al., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, Austin Tex.) and Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997.
CAD-mediated cell adhesion triggers a cascade of events that lead to the formation of intercellular junctions, and ultimately to the establishment of permeability barriers between tissue compartments. The intercellular junction that is directly responsible for the creation of permeability barriers that prevent the diffusion of solutes through paracellular spaces is known as the tight junction, or zonula occludens (Anderson and van Itallie, Am. J. Physiol. 269:G467-G475, 1995; Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997).
Occludin is a transmembrane component of tight junctions (Furuse et al., J. Cell Biol. 123:1777-1788, 1993; Furuse et al., J. Cell Sci. 109:429-435, 1996). This protein appears to be expressed by all endothelial cell types, as well as by most epithelial cell types. Occludin is an integral membrane protein (FIG. 1) that is composed of two extracellular domains (EC1 and EC2), four hydrophobic domains (TM1-TM4) that transverse the plasma membrane, and three cytoplasmic domains (CP1-CP3). The structures of all known mammalian occludins are similar (FIG. 2; Ando-Akatsuka et al., J. Biol. Chem. 133:43-47, 1996). Occludin is believed to be directly involved in cell adhesion and the formation of tight junctions (Furuse et al., J. Cell Sci. 109:429-435, 1996; Chen et al., J. Cell Biol. 138:891-899, 1997). It has been proposed that occludin promotes cell adhesion through homophilic interactions (an occludin on the surface of one cell binds to an identical occludin on the surface of another cell). A detailed discussion of occludin structure and function is provided by Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997.
Although cell adhesion is required for certain normal physiological functions, there are situations in which the level of cell adhesion is undesirable. For example, many pathologies (such as autoimmune diseases and inflammatory diseases) involve abnormal cellular adhesion. Cell adhesion may also play a role in graft rejection. In such circumstances, modulation of cell adhesion may be desirable.
In addition, permeability barriers arising from cell adhesion create difficulties for the delivery of drugs to specific tissues and tumors within the body. For example, skin patches are a convenient tool for administering drugs through the skin. However, the use of skin patches has been limited to small, hydrophobic molecules because of the epithelial and endothelial cell barriers. Similarly, endothelial cells render the blood capillaries largely impermeable to drugs, and the blood/brain barrier has hampered the targeting of drugs to the central nervous system. In addition, many solid tumors develop internal barriers that limit the delivery of anti-tumor drugs and antibodies to inner cells.
Attempts to facilitate the passage of drugs across such barriers generally rely on specific receptors or carrier proteins that transport molecules across barriers in vivo. However, such methods are often inefficient, due to low endogenous transport rates or to the poor functioning of a carrier protein with drugs. While improved efficiency has been achieved using a variety of chemical agents that disrupt cell adhesion, such agents are typically associated with undesirable side-effects, may require invasive procedures for administration and may result in irreversible effects.
Further, internal barriers developed by tumors inhibit the immune system""s ability to attack tumor cells. Immune cells such as leukocytes often cannot infiltrate a tumor, and thus tumor cells are protected from the body""s natural defenses. There are presently no available methods for enhancing immune cell infiltration of solid tumors.
Accordingly, there is a need in the art for compounds that modulate cell adhesion, improve drug delivery across permeability barriers and permit immune cell infiltration of solid tumors. The present invention fulfills this need and further provides other related advantages.
The present invention provides compounds and methods for modulating occludin-mediated cell adhesion and the formation of permeability barriers. Within certain aspects, compounds provided herein comprise an occludin CAR sequence, or variant thereof that retains the ability to modulate occludin-mediated cell adhesion. Certain compounds are cyclic peptides that comprise the sequence LYHY (SEQ ID NO:1). Within certain embodiments, such cyclic peptides have the formula: 
wherein X1, and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; wherein Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and wherein Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds. Such cyclic peptides may comprise modifications such as an N-acetyl or N-alkoxybenzyl group and/or a C-terminal amide or ester group. Cyclic peptides may be cyclized via, for example, a disulfide bond; an amide bond between terminal functional groups, between residue side-chains or between one terminal functional group and one residue side chain; a thioether bond or xcex41xcex41-ditryptophan, or a derivative thereof.
Within other embodiments, such compounds may be linear peptides comprising the sequence LYHY (SEQ ID NO:1) or a variant thereof. Such peptides are preferably 4-30 amino acid residues in length, preferably 5-16 amino acid residues, and more preferably 6-9 amino acid residues.
Within further aspects, the present invention provides cell adhesion modulating agents that comprise a cyclic or linear peptide as described above. Within specific embodiments, such modulating agents may be linked to one or more of a targeting agent, a drug, a solid support or support molecule, or a detectable marker. Within further specific embodiments, cell adhesion modulating agents are provided that comprise a sequence selected from the group consisting of QYLYHYCVVD (SEQ ID NO:2), YLYHYCVVD (SEQ ID NO:12), LYHYCVVD (SEQ ID NO:13), QYLYHYC (SEQ ID NO:14), YLYHYC (SEQ ID NO:15), LYHYC (SEQ ID NO:16), QYLYHY (SEQ ID NO:17), YLYHY (SEQ ID NO:18) and derivatives of the foregoing sequences having one or more C-terminal, N-terminal and/or side chain modifications.
Within further related aspects, cell adhesion modulating agents are provided which comprise an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an occludin.
In addition, any of the above cell adhesion modulating agents may further comprise one or more of: (a) a cell adhesion recognition sequence that is bound by an adhesion molecule other than an occludin, wherein said cell adhesion recognition sequence is separated from any LYHY (SEQ ID NO:1) sequence(s) by a linker; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than an occludin.
The present invention further provides pharmaceutical compositions comprising a cell adhesion modulating agent as described above, in combination with a pharmaceutically acceptable carrier. Such compositions may further comprise a drug. In addition, or alternatively, such compositions may further comprise one or more of: (a) a peptide comprising a cell adhesion recognition sequence that is bound by an adhesion molecule other than an occludin; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than an occludin.
Within further aspects, methods are provided for modulating cell adhesion, comprising contacting a cadherin-expressing cell with a cell adhesion modulating agent as described above.
The present invention further provides methods for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above and a drug, wherein the modulating agent inhibits occludin-mediated cell adhesion.
Within further aspects, the present invention provides methods for treating cancer in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits occludin-mediated cell adhesion.
Within further aspects, the present invention provides methods for enhancing immune cell infiltration of a tumor in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits occludin-mediated cell adhesion.
These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually noted for incorporation.